There are many methodological issues to consider when assessing adult neurogenesis in vivo.
One issue is the method of identifying proliferating NSCs and their daughter cells. The earliest studies used [3H]-thymidine, which gets incorporated into the DNA during the S-phase of the cell cycle, labels dividing cells and their progeny in vivo and can be detected by autoradiography.
The introduction of the synthetic thymidine analogue 5-bromo-2'deoxyuridine (BrdU) substitutes for thymidine in newly synthetized DNA of proliferating cells. BrdU incorporated into DNA can then be easily visualized by immunohistochemistry using specific anti-BrdU antibodies. With this method it is possible to perform a quantitative analysis of proliferation, phenotypic differentiation, and survival of newborn cells by varying the time interval between the pulsed administration of BrdU and the perfusion of animals. Using this approach, one can estimate the number of cells in S phase at a particular time if brains are examined at a relatively short time (e.g. 1 hour) after a BrdU pulse; proliferating cells can be tracked through several anatomical divisions, when examined several days or weeks after a BrdU pulse. Even though BrdU labeling is considered the gold standard for detection of adult neurogenesis, it has several pitfalls. To provide unambiguous data for neurogenesis, one must demonstrate BrdU-co-localisation with phenotypic markers in three orthogonal planes using confocal microscopy. Additionally, sources of false positive phenomena need to be excluded (mitosis-like apoptosis, DNA repair, fusion of neurons with proliferating glia, DNA endo-replication). Although DNA labeling by BrdU is currently the most commonly used method for studying adult neurogenesis, the potential toxic effect of this thymidine analogue should not be ignored, as it might be a confounding factor in some experiments.
The introduction of the synthetic thymidine analogue 5-bromo-2'deoxyuridine (BrdU) substitutes for thymidine in newly synthetized DNA of proliferating cells. BrdU incorporated into DNA can then be easily visualized by immunohistochemistry using specific anti-BrdU antibodies. With this method it is possible to perform a quantitative analysis of proliferation, phenotypic differentiation, and survival of newborn cells by varying the time interval between the pulsed administration of BrdU and the perfusion of animals. Using this approach, one can estimate the number of cells in S phase at a particular time if brains are examined at a relatively short time (e.g. 1 hour) after a BrdU pulse; proliferating cells can be tracked through several anatomical divisions, when examined several days or weeks after a BrdU pulse. Even though BrdU labeling is considered the gold standard for detection of adult neurogenesis, it has several pitfalls. To provide unambiguous data for neurogenesis, one must demonstrate BrdU-co-localisation with phenotypic markers in three orthogonal planes using confocal microscopy. Additionally, sources of false positive phenomena need to be excluded (mitosis-like apoptosis, DNA repair, fusion of neurons with proliferating glia, DNA endo-replication). Although DNA labeling by BrdU is currently the most commonly used method for studying adult neurogenesis, the potential toxic effect of this thymidine analogue should not be ignored, as it might be a confounding factor in some experiments.
This has led to the use of other markers of the cell cycle to analyse cell proliferation in situ, such as proliferating nuclear antigen (PCNA) and Ki-67. However, since the expression of these markers is turned off after the termination of the mitotic cell cycle, the phenotypic differentiation of the newborn daughter cells can not be tracked.
To overcome this drawback, retroviral transfection may be used for labeling mitotic cells by forced expression of green fluorescent protein (GFP) or other reporter genes, thus allowing a complete morphological or electrophysiological analysis of the daughter cells (see video, unpublished data).
A second methodological issue concerns criteria for unambiguous identification of a cellular phenotype. Presently, the neuronal nuclei antigen (NeuN) is the standard immnunocytochemical marker to identify mature neurons. Glial fibrillary acidic protein (GFAP) and S100β are used as markers for mature astrocytes. However, at present there are no undisputed marker proteins that can provide unambiguous prospective identification of intermediate precursor cells in the adult brain.
References
Arias-Carrión O, Yamada E, Freundlieb N, Djufri M, Maurer L, Hermanns G, Ipach B, Chiu WH, Steiner C, Oertel WH, Höglinger GU. Neurogenesis in substantia nigra of Parkinsonian brains? J Neural Transm Suppl 73:279-85 (2009)
Depboylu C, Schäfer MKH, Arias-Carrión O, Oertel WH, Weihe E, Höglinger GU. Possible Involvement of complement factor C1q in the clearance of extracellular neuromelanin from the substantia nigra in Parkinson’s disease. J Neuropathol Exp Neurol 70 (2) 125-132 (2011)
Arias-Carrión O, Freundlieb N, Oertel WH, Höglinger GU. Adult Neurogenesis and Parkinson’s disease. CNS Neurol Disord Drug Targets 6:326-35 (2007)
Arias-Carrión O, Drucker-Colín R. Neurogenesis as a therapeutic strategy to regenerate central nervous system. Rev Neurol 45: 739-745 (2007)
Arias-Carrión O. Basic mechanisms of rTMS: Implications in Parkinson's disease. Int Arch Med 1(1):2 (2008)
Yuan TF, Arias-Carrión O. Adult neurogenesis in the hypothalamus: Evidences, functions and implications. CNS Neurol Disord Drug Targets 10(4):433-439 (2011)
